Method for the determination of sarcosine creatine or creatinine

ABSTRACT

The present invention provides a method of use for a hydrogen peroxide-forming oxidase, wherein the enzyme is obtained from Streptomycetaceae and at 25° C., in 0.15 mol/liter potassium phosphate (pH 7.9), in the presence of surface-active substances, still shows after 2 days an activity of at least 40% of the initial activity. This enzyme is useful in the determination of sarcosine, creatine and creatinine.

This is a divisional application of U.S. Ser. No. 868,262 filed May 28, 1986 now U.S. Pat. No. 4,743,549.

The present invention is concerned with a new sarcosine oxidase with improved stability in comparison with known sarcosine oxidases, especially in detergent-containing analysis reagents.

Sarcosine oxidases (E.C. 1.5.3.1) can be used, inter alia, for the enzymatic determination of sarcosine, creatine and creatinine, the enzymatic determination of creatinine in serum, plasma or urine being of especial importance in clinical diagnosis. By coupling of the reactions catalysed by creatinine amidohydrolase (E.C. 3.5.2.10), creatine amidinohydrolase (E.C. 3.5.3.3) and sarcosine oxidase, hydrogen peroxide is finally formed from creatinine in the stoichiometric ratio of 1:1 and this can be determined colorimetrically in a simple way. Numerous chromogenic systems have proved to be especially useful for this colorimetric determination, for example those of the Trinder type (see Bergmeyer "Methoden der enzymatischen Analyse", 4th edition, Volume 1 (1983) page 197), in the case of which, in the presence of peroxidase, from 4-aminoantipyrine and a phenolic or anilinic coupler, a coloured material is produced oxidatively by hydrogen peroxide, the amount (or intensity) of this coloured material being in linear relationship to the amount of hydrogen peroxide formed.

Such a creatinine detection based upon the sarcosine reaction offers, in comparison with known enzymatic creatinine tests (Bergmeyer, "Methoden der enzymatischen Analyse", 3rd edition, Volume II (1974) page 1834, Tanganelli et al., Clin. Che., 28, 1461/1983), the advantage, depending upon the nature of the colour coupler used, of a distinctly higher detection sensitivity which, precisely because of the low serum creatinine concentration in the diagnostically decisive range (44-97 μmol/1.), is of great importance for the exactitude of the anlysis. In addition, the stability of the chromogenic substances in the neutral, aqueous medium employed is also better than that of NADH, the indicator used in UV tests. Also in comparison with the routine method even today used the most frequently, the creatinine determination according to Jaffe (Hoppe-Seyler's Z. Physiol. Chem., 10, 391/1886), this process also offers a substantially greater specificity and thus an improved diagnostic dependability. Finally, the use of corrosive, strongly alkaline reagents is thereby also avoided.

However, the use of sarcosine oxidase, for example for the determination of creatinine, requires that, in the regent ready for use, it is sufficiently storage-stable for at least several days at 0° to 25° C. and, furthermore, in the case of carrying out the measurement even at elevated temperatures (30° to 37° C.), no significant loss of activity occurs over the minimum reaction time necessary. Since, in clinical chemistry, turbid, triglyceride-rich sera are frequently obtained as sample material, enzymatic analysis reagents preferably also contain so called clarification systems, most of which consist of a combination of lipases with non-ionic detergents (polyoxyethylated alkyl or aralkyl alcohols) and salts of bile acids, such as sodium cholate, as solubilising agent which permits the disturbance-free optical measurement even of strongly lipaemic samples.

In order, for example, also to be able to carry out the creatinine determination in such turbid samples, it is, therefore, necessary that the sarcosine oxidase, under the above-mentioned storage and reaction conditions, is sufficiently resistant towards denaturing or inactivation by detergents.

Furthermore, it is desirable that the sarcosine oxidase has appropriate enzymatic properties, for example a low Michaelis constant for sarcosine and a high maximum reaction rate, since, due to these properties, there is essentially co-determined the reaction time in the case of sarcosine, creatine and creatinine determinations. Since the enzymatic determination of, for example, creatinine is also to be capable of being carried out at higher temperatures (37° C.) and in the presence of detergents and solubilisers, appropriate enzymatic properties are of considerable importance in the case of these stressing ambient conditions.

Corresponding investigations showed that the known sarcosine oxidases in such detergent-containing analysis reagents do not display a storage stability satisfying these requirements and/or a stability at elevated reaction temperatures.

Therefore, there is a need for a sarcosine oxidase which possesses the above-mentioned properties and especially fulfills the stability criteria.

Thus, according to the present invention, there is provided a sarcosine oxidase obtainable from Streptomycetaceae which, at 25° C. in 0.15 mol/liter potassium phosphate (pH 7.9) and in the presence of surface-active substances, still shows after 2 days an activity of at least 40% of the initial activity.

At 37° C. in a detergent-containing medium, the sarcosine oxidase according to the present invention possesses for sarcosine a K_(M) value of 2 to 4 mmol/liter. In contradistinction thereto, the K_(M) values of the known sarcosine oxidases which are sufficiently stable for these determinations are, under these conditions, abou 16 to 20 mmol/liter.

The enzyme according to the present invention is found in all species of the family Streptomycetacea (The Prokaryotes, Vol. II (1981), 2028), for example in Chainia purpurogena DSM 43 156, Chainia ochraceae DSM 43 155, Streptomyces fluocculus DSM 40 327, Streptoverticillium sp. DSM 40 237) and Kitasatoa purpurea DSM 43 362.

The enzyme of the present invention is composed of four different sub-units and its molecular weight is about 170 kD.

The enzyme is stable in the pH range of 6 to 9 and at temperatures below 40° C. At 50° C., it is inactivated within 15 minutes. The optimum temperature of the reaction is about 37° C. and the pH optimum is pH 8.0. The high substrate specificity is shown by the very low conversion of substrate analogues; thus, the conversion rate of, for example, N,N-dimethylglycine is only 1% of that of the sarcosine-specific reaction.

The K_(M) values for sarcosine (phosphate buffer; TES buffer), measured at 25° C. in various stressing reagents, are 2 to 3 mmol/litre. The V_(max) is about 6 U/mg. protein. The followig Table I gives, for various preparations of the enzyme according to the present invention, the K_(M) values for sarcosine measured at 25 and at 37° C. For comparison, there are given the corresponding values for the known Bacillus enzyme.

                  TABLE I                                                          ______________________________________                                                                         K.sub.M value                                                                  (mmol/l) for                                                                   sarcosine in                                   origin of the sarcosine                                                                     T                  stressing                                      oxidase      (°C.)                                                                            buffer    reagent a                                      ______________________________________                                         Chainia purpurogena                                                            (DSM 43 156)                                                                   pure enzyme  25       phosphate 2                                              pure enzyme  37       phosphate 3.5                                            pure enzyme  25       TES       2.8                                            pure enzyme  37       TES       3                                              Chainia purpurogena                                                            crude extract                                                                  supernatant  37       phosphate 3                                              supernatant  37       TES       3                                              Chainia ochraceae                                                              (DSM 43 155)                                                                   crude extract                                                                  supernatant  37       phosphate 3.5                                            supernatant  37       TES       3.6                                            Streptomyces flocculus                                                         (DSM 40 327)                                                                   crude extract                                                                  supernatant  37       TES       3.5                                            Bacillus sp. 25       phosphate 16                                             Bacillus sp. 25       TES       20                                             Bacillus sp. 37       phosphate 18                                             Bacillus sp. 37       TES       20                                             ______________________________________                                    

Stressing reagent a:

0.15 mole potassium phosphate or 0.1 mole TES/KOH (pH 7.9), 8.6 mmole 2,4,6-tribromo-3-hydroxybenzoic acid, 0.8 mmole 4-aminoantipyrine, 10 μmole potassium ferrocyanide, 5 mmole sodium cholate, 0.5% Lutensol ON 50, 0.2% sodium azide, 0.5 mmole Titriplex III, 2000 U lipase, 2000 U peroxidase and 10000 U ascorbate oxidase, per liter. [Lutensol ON 50 ^(R) is obtained from BASF AG and is n-decanol-polyglycolether having an average of 5 ethylene glycol units per molecule i.e. CH₃ -(CH₂)₉ -(OCH₂ -CH₂)₅ -OH. Titriplex III is the disodium salt of ethylenediaminetetracetic acid and can be obtained from Merck.]

The enzyme according to the present invention has a superior stability in a detergent-containing medium at 37° C. which is shown not only by the crude extract supernatant but also by the purified enzyme. The following Table II shows the stability of the enzyme according to the present invention in comparison with known sarcosine oxidases.

                  TABLE II                                                         ______________________________________                                                       % residual activity after                                                      incubation in stressing                                                        reagent b at 37° C. over                                  origin of the sarcosine                                                                      different time intervals                                         oxidase       15 min.  30 min.  45 min.                                                                               60 min.                                 ______________________________________                                         Pseudomonas maltophilia                                                                      7        3        2      0                                       Corynebacterium sp.U96                                                                       6        2        1      0                                       Bacillus sp.  87       84       78     75                                      Arthrobacter sp.                                                                             7        1        0      0                                       Cylindrocarpon                                                                               0        0        0      0                                       didymum M-1                                                                    Chainia purpurogena                                                                          90       87       83     77                                      pure enzyme                                                                    Chainia purpurogena                                                                          90       87       82     75                                      crude extract                                                                  supernatant                                                                    Streptomyces flocculus                                                                       97       97       96     94                                      crude extract                                                                  supernatant                                                                    ______________________________________                                    

Stressing reagent b:

0.15 mole potassium phosphate (pH 7.9), 8.6 mmole 2,4,6-tribromo-3-hydroxybenzoic acid, 0.8 mmole 4-aminoantipyrine, 10 μmole potassium ferrocyanide, 5 mmole sodium chlate, 0.5% Lutensol ON 50, 0.2% sodium azide, 0.5 mmole Titriplex III, 2000 U lipase, 2000 U peroxidase, 10000 U ascorbate oxidase, 25000 U creatininase, 12000 U creatinase, >100 U sarcosine oxidase.

The above values show that only the enzyme from Bacillus displays a comparable stability, whereas all other enzymes possess a stability which is fully insufficient for practical use.

The following Table III shows the long-term stability at 25° C. of the enzyme according to the present invention and of the Bacillus sp. enzyme.

                  TABLE III                                                        ______________________________________                                         origin of              % residual activity after                               the sarcosine          two days incubation in                                  oxidase     buffer     stressing reagent c at 25° C.                    ______________________________________                                         Chainia purp.                                                                              phosphate  83                                                      pure enzyme TES        80                                                      Chainia purp.                                                                              phosphate  86                                                      crude extract                                                                              TES        88                                                      Chainia ochr.                                                                              phosphate  75                                                      crude extract                                                                              TES        75                                                      Streptomyces                                                                               phosphate  44                                                      flocc.                                                                         crude extract                                                                              TES        35                                                      Bac. sp.    phosphate  15                                                      pure enzyme TES        15                                                      ______________________________________                                    

Stressing reagent c:

Composition as for stressing agent b but without the addition of the chromogenic colour system. Besides 0.15 mole potassium phosphate (pH 7.9), 0.1 mole TES/KOH is also used.

The above values show that the enzyme of the present invention is far superior to the best previously known sarcosine oxidase enzyme with regard to long-term stability. This is especially important for the storage stability which correlates with the long-term stability.

Therefore, because of its smaller Michaelis constant, the enzyme according to the present invention makes possible a substantially quicker carrying out of the enzymatic determination of sarcosine, creatine or creatinine. It has a very substantially better storage stability at 0° to 25° C. and, over the incubation interval, is, in the case of sarcosine, creatine or creatinine determinations at 37° C., substantially more stable than most of the known sarcosine oxidases.

The following Examples are given for the purpose of illustrating the present invention:

EXAMPLE 1 Culturing of Chainia Purpurogena DSM 43156

The organism was cultured in a complex medium of the following composition in a shaking flask: 5 g. yeast extract, 3 g. peptone (tryptic digested), 2 g. sodium chloride, 0.24 g. magnesium sulphate heptahydrate, 0.014 g. calcium chloride heptahydrate, 2 g. glucose, 10 g. sarcosine and 1 liter water (pH 7.0). The cultures produced at 28° C. yielded, after 30 hours, an activity of about 400 U/liter.

EXAMPLE 2 Isolation of Sarcosine Oxidase (E.C. 1.5.3.1) from Chainia

2.9 kg. of moist mass of Chainia purpurogena DSM 43156 (obtained from 95 liters of culture) were suspended in 15 liters of 20 mmole/litre phosphate buffer (pH 8.0) and digested for 4 hours at 25° C. with 4 g. lysozyme. To the digestion suspension was added so much 10% polyethyleneimine (Polymin G-20) solution (BASF) that a substantial separation of nucleic acids and foreign proteins took place. The carsocine oxidase, which was present in the supernatant, was bound to a weakly basic anion exchanger (DEAE-Sephadex) and subsequently eluted with increasing salt gradients. The eluate was adjusted with ammonium sulphate to a concentration of 0.6 mole/liter and the enzyme was bound to phenyl-Sepharose and chromatographed with decreasing ammonium sulphate gradients (above phosphate buffer). The eluates with over 4 U/mg. of protein were adjusted with ammonium sulphate to a concentration of up to 2.4 mole/liter. The precipitate was taken up in 0.1 mole/liter phosphate buffer and the sarcosine oxidase further purified by passage over a molecular sieve (sephacryl-S-200, Pharmacia). The purified enzyme obtained had a specific activity of 5.5 U/mg. protein.

EXAMPLE 3 Use of Sarcosine Oxidase for the Determination of Creatinine

(a) Reagent I (sample blank reagent)

    ______________________________________                                         potassium phosphate (pH 7.9)                                                                      150 mmole/1.                                                                   (or 0.1 mole/1. TES/KOH                                                        pH 7.9)                                                     4-aminoantipyrine  0.8 mmole/1.                                                2,4,6-tribromo-3-hydroxy-                                                      benzoic acid       8.6 mmole/1.                                                potassium ferrocyanide                                                                            10 μmole/1.                                              sodium cholate     5 mmole/1.                                                  Lutensol ON 50     0.5% (w/v)                                                  creatinamidinohydrolase                                                                           12 U/ml.                                                    sarcosine oxidase according                                                                       6.5 U/ml.                                                   to Example 2                                                                   peroxidase         2 U/ml.                                                     lipase             2 U/ml.                                                     ascorbate oxidase  10 U/ml.                                                    ______________________________________                                    

(b) Reagent II (sample reagent)

Reagent I plus creatinin-amidohydrolase: 25 U/ml.

(c) Carrying out of test/determination batch

wavelength 546 nm; T=25° C. (or 37° C.); layer thickness =1 cm., measurement against air.

    ______________________________________                                                1.      2.        3.        4.                                                 Reagent I                                                                              sample    Reagent II                                                                               sample                                             blank   blank     blank     value                                       ______________________________________                                         Reagent I                                                                               1.00 ml.  1.00 ml.    --    --                                        Reagent II                                                                              --        --          1.00 ml.                                                                             1.00 ml.                                  water    0.05 ml.  --          0.05 ml.                                                                             --                                        sample   --        0.05 ml.    --    0.05 ml.                                  ______________________________________                                    

Incubate for 20 minutes at 25° or 37° C., then measure extinctions E₁ E₄. E=(E₄ -E₃)-(E₂ -E₁). Calculation of the creatinine concentration in the sample: via concurrently conducted aqueous standard (2 mg./dl.). The standard is, for this purpose, introduced into the determination batch in the same way as the sample. 

We claim:
 1. A method for the determination of sarcosine which comprises:(1) obtaining hydrogen peroxide-forming sarcosine oxidase, from Streptomycetaceae wherein said enzyme at 25° C. in 0.15 mol/liter potassium phosphate (pH 7.9), in the presence of surface-active substances, still shows after 2 days an activity of at least 40% of the initial activity; (2) reacting a sample containing sarcosine with the sarcosine oxidase obtained in step 1), and (3) measuring the H₂ O₂ produced.
 2. Method for the determination of creatine which comprises:(1) obtaining hydrogen peroxide-forming sarcosine oxidase, from streptomycetaceae wherein said enzyme 25° C. in 0.15 mol/litre potassium phosphate (pH 7.9), in the presence of surface-active substances, still shows after 2 days an activity of at least 40 of the initial activity; (2) reacting a sample containing creatine with creatineamidinohydrolase whereby sarcosine and urea are formed and further reacting the sarcosine with the sarcosine oxidase obtained in step 1, and (3) measuring the H₂ O₂ produced.
 3. Method for the determination of creatinine which comprises:(1) obtaining hydrogen peroxide-forming sarcosine oxidase, from Streptomycetaceae wherein said enzyme at 25° C. in 0.155 mol/litre potassium phosphate (pH 7.9), in the presence of surface-active substances, still shows after 2 days an activity of at least 40% of the initial activity; (2) reacting creatinine with creatininamidohydrolase to form creatine, reacting the so-formed creatine with creatinamidinohydrolase to form sarcosine and urea, andn further reacting the sarcosine with the sarcosine oxidase obtained in step 1, and (3) measuring the H₂ O₂ produced.
 4. Method of claim 1, 2 or 3 wherein the reaction takes place in stressing medium.
 5. Method of claim 1, 2 or 3 wherein the reaction takes place in a clinical diagnosis using body fluids.
 6. Method of claim 1, 2 or 3 wherein the sarcosine oxidase is obtained from the species of Streptomycetaceae selected from the group consisting of Chainia purpurogena, Chainia ochraceae, Streptomyces flocculus, Streptoverticilliumand nKitasatoa purpurea.
 7. Method of claim 5 wherein the body fluids are serum, plasma or urine.
 8. Method of claim 2 or 3 wherein the H₂ O₂ produced is determined coloremetrically. 